GSM8084186: ChIP-SMAD2/3_SB; Mus musculus; ChIP-Seq
For ChIP, 1x107 cells were crosslinked at room temperature for 10 min by swirling with 1% formaldehyde (Sigma Aldrich) followed by addition of 125 mM glycine to quench the reaction. After washing the cells three times with PBS, cells were resuspended with ChIP lysis buffer (50 mM HEPES/KOH pH 7.5, 150 mM NaCl, 0.1% Na-deoxycholate, 1% Triton X-100, 1 mM EDTA, 0.1% SDS) supplemented with protease inhibitors (Roche, cOmplete, mini, EDTA-free protease inhibitor tablets, Cat #: 11836170001) and phosphatase inhibitors (Thermo Scientific, Halt Phosphatase Inhibitor Cocktail, Cat #: 78427, 1:1000). Cell lysates in 1 mL of ChIP lysis buffer were sonicated for sheared chromatin to be 200-500 bp. Supernatants cleared by centrifugation were incubated with the designated antibody at 4ºC overnight on a rotator. Dynabeads™ Protein G were blocked with 2.5 mg/mL UltraPure™ BSA (Thermo Scientific, Cat #: AM2616) at 4ºC overnight. The beads were then washed three times with ChIP buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1mM EDTA, 1% Triton X-100) and incubated with the chromatin-antibody samples at 4ºC for 1-2 h. These samples were washed 6 times with high salt ChIP buffer (20 mM Tris, pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% of Triton X-100, 0.1% of SDS) followed by two washings with Tris-EDTA (TE) buffer. Chromatin complexes were eluted twice in TE with 1% ultrapure SDS (Thermo Fisher Scientific) at 65ºC for 15 min, and reverse-crosslinked with RNAse A (Thermo Fisher Scientific, Cat #: EN0531) for 4 h or overnight, followed by incubation with Proteinase K solution (Thermo Fisher Scientific, Cat #: EO0491) for 1 h at 45ºC. DNA was purified using QIAquick PCR Purification Kit (Qiagen). For CUT&RUN, samples were prepared with CUTANA™ ChIC/CUT&RUN Kit (Epicypher, Cat #: 14-1048). For each CUT&RUN reaction, cell pellets (5x105 cells) were washed twice with wash buffer (20 mM HEPES–KOH, pH 8.0; 150 mM NaCl; 0.5mM spermidine; and protease inhibitor (Roche), followed by resuspension with wash buffer. Cells in wash buffer were incubated with activated Concanavalin-A beads for 10 min at room temperature. Beads were resuspended with immunoprecipitation buffer (Wash buffer; 0.01% digitonin; 2 mM EDTA) and incubated with 0.5 µg of designated antibody at 4ºC overnight on a nutator. The following day, beads were washed twice with digitonin buffer (wash buffer including 0.01% digitonin) and incubated with CUTANA pAG-MNase for 10 min at room temperature, followed by wash twice with digitonin buffer. pAG-MNase was activated by incubation with 1 µL of 100 mM CaCl2 for 2 h at 4ºC on nutator. The reaction was terminated by incubation with stop buffer for 10 min at 37 ºC in thermocycler. CUT&RUN enriched DNA was purified using CUTANA DNA purification kit (Epicypher, Cat #: 14-0050). Immunoprecipitated DNA was quantified by PicoGreen and the size was evaluated by Agilent BioAnalyzer. Illumina sequencing libraries were prepared using the KAPA HTP Library Preparation Kit (Kapa Biosystems, Cat #: KK8234) according to the manufacturer's instructions with 0.02-5 ng input DNA and 8-14 cycles of PCR. Barcoded libraries were run on the NovaSeq 6000 in a PE100 run, using the NovaSeq 6000 S2 or S4 Reagent Kit (200 or 300 Cycles) (Illumina).